The dynamics of the immune system have an enormous impact on an individual""s health and well-being. T cells, the regulators of immune response, have a particularly important role, in determining when, or if, to mount a response against a particular antigen. Inappropriate T cell response to self-antigens can be deadly, and so several mechanisms serve to establish tolerance. These are broadly grouped into central; or peripheral tolerance.
Unlike central tolerance, in which auto-reactive T cell are deleted; peripheral tolerance involves induction of an unresponsive state, termed anergy. The anergic state is induced through a partial activation process (Harding et al. (1992) Nature 370:607-609). When peripheral T cells encounter antigen, they are normally presented with two molecular signals. The first signal is binding of the T cell antigen receptor to a peptide antigen presented by an MHC class II molecule. The second, or xe2x80x9ccostimulatoryxe2x80x9d signal, is provided by interaction of B7 like molecules on the antigen presenting cell (APC) with the CD28 receptor on the T cell. Antigen stimulation in the absence of sufficient costimulatory signals results in anergy. Anergic T cells are characterized by greatly reduced or absent IL-2 production, and a lack of proliferation in response to full activation.
There is a therapeutic interest in understanding the mechanisms that underlie anergy. Loss of anergy in T cells that recognize self-antigens can lead to autoimmune diseases such as insulin dependent diabetes, rheumatoid arthritis, and multiple sclerosis. In one example of a therapeutic use, the anergy inducing molecule CTLA4Ig has been tested in clinical trials for the treatment of the autoimmune disease psoriasis vulgaris. Conversely, inappropriate anergy may be associated with cancer, where the body fails to mount a response to tumor antigens.
Although it has been well established that TCR signaling in the absence of CD28 costimulation leads to T cell anergy, the actual mechanism(s) of anergy induction are not well characterized. It has been shown, for example, that anergized T cells exhibit a blockade in intracellular signaling pathways leading to IL-2 gene transcription, which may be attributed to diminished nuclear translocation of the transcription factor AP-1, Kang et al. (1992) Science 257:1134-1138). It has also been demonstrated that the Ras signaling pathway, which ultimately leads to AP-1 translocation, may be xe2x80x9cdefectivexe2x80x9d in anergized T cells (Fields et al. (1996) Science 271:1276-1278). While these studies suggested that blockade of certain signaling pathways was involved in the functional state of anergy, they did not address whether early changes in gene expression were involved in anergy induction. The further identification and evaluation of genes involved in the induction and maintenance is therefore of great clinical and scientific interest.
Relevant Literature
One tool showing considerable promise for expression analysis is the nucleic acid array, reviewed by Ramsay (1998) Nat. Biotech. 16:40-44. These arrays contain dense collections of nucleic acids, either PCR products or oligonucleotides, usually of known sequence, that have been either synthesized or printed at fixed spatial locations on suitable substrates, such as nylon filters or glass slides. When labeled DNA or RNA samples are hybridized to the arrays, the abundance of specific sequences in solution can be quantitated based on the fluorescent or radioactive signal intensity at the position of the complementary probe. A number of methods are available for creating microarrays of biological samples, exemplary are PCT Application Ser. No. WO95/35505, published Dec. 28, 1995; U.S. Pat. No. 5,445,934, issued Aug. 29, 1995; and Drmanac et al., Science 260:1649-1652.
Changes in gene expression related to anergy have been explored in the literature. Bousslotis et al. (2000) Nat Med 6(3):290-7 report that p27kip1 cyclin-dependent kinase inhibitor is involved in the blockade of clonal expansion of anergic T cells. Korthauer et al. (2000) J Immunol 164(1):308-18 used differential display of reverse transcribed RNA to identify genes selectively induced in anergic T cells. Powell et al. (1998) Immunol Rev 165:287-300 report that T-cell anergy appears to be an active negative state in which IL-2 production is inhibited both at the level of signal transduction and by cis-dominant repression at the level of the IL-2 promoter.
Hautamaa et al. (1997) Cytokine 9(6):375-82 used a differential screening approach to clone murine lymphotactin from a cDNA library produced from an unresponsive Th1 cell.
Isolated nucleic acid compositions and sequences of anergy associated genes are provided, including the novel GRAIL gene. Expression of these genes is upregulated during the early stages of induction of anergy. The nucleic acid compositions find use in identifying homologous or related genes; in producing compositions that modulate induction or maintenance of anergy; for gene therapy; mapping functional regions of the encoded protein; and in studying associated physiological pathways. In addition, modulation of the gene activity in vivo is used for prophylactic and therapeutic purposes, such as treatment of autoimmune disease, identification of anergic T cells, and the like. The GRAIL sequence is shown to attenuate IL-2 transcription in T cells during; response to antigenic stimulation.
The identification of genes involved in the induction of anergy is useful in the evaluation of the pathophysiology or immunotherapy of cancer, autoimmune disease, and transplant rejection. Genetic sequences involved in anergy induction are useful markers in the evaluation of specific immunotherapies. Functional characterization of genes involved in anergy induction allows the elucidation of the mechanism(s) of T cell anergy, including the transcriptional blockade of IL-2, which may be manipulated to regulate T cell responses in human disease.